Membrane nanodiscs: a platform for the structural and functional characterization of a bacterial PIB-type ATPase

Introduction:

Reconstitution of membrane proteins in a native-like lipidic environment is vital for the determination of their structural and functional properties. Nanodics (NDs) constitute such an environment by providing a planar nanoscale phospholipid bilayer bounded by a ring of two membrane scaffolding proteins (MSP). NDs are advantageous over liposomes and bicelles as they exhibit less scatter, are soluble and of defined size and can integrate membrane proteins as single molecules that are accessible from both sides. Here we show that NDs can be utilized to study the mechanism of PIB-type ATPases, a protein family pumping transition metals, e.g. copper, across biological membranes. Moreover, we explore the potential of ND for proposed single molecule structural studies using X-FEL radiation.

Objectives:

We want to explore the use of NDs as a biochemical platform for spectroscopic, biochemical and ultimately single molecule structural studies of membrane proteins, as for example the allosteric coupling between copper transport and ATP-hydrolysis in the PIB-type ATPase CopA from L. pneumophila.

Materials and Methods:
CopA and MSP1 were affinity-purified following recombinant expression in E. coli. Nanodiscs were reconstituted from a MSP : lipid : CopA (1:60:0.1) cholate-mixture after detergent removal and characterized by dynamic light scattering, AFM, CD spectroscopy and X-ray diffraction using X-FEL radiation.

Results:

MSP1 was purified and NDs were prepared with different lipid moieties. Thorough analyses by natural and magnetic CD as well as dynamic light scattering show a well defined and stable composition of NDs of an average diameter of 10nm. Preliminary x-ray diffraction data from NDs have been recorded using the X-FEL at SLAC, (USA).
After successfull purification of CopA from L. pneumophila, enzyme activity was tested by ATPase assays and spectroscopic techniques, followed by reconstitution into NDs for further biophysical investigation.

Conclusions: NDs provide a suitable platform for structural and functional studies of integral membrane proteins in a native-like lipidic environment. They offer the potential to apply functional assays under identical conditions as envisaged advanced x-ray diffraction experiments using pulsed r-ray sources. Here, we have established the functional reconstitution of the bacterial heavy-metal pump CopA into NDs to study structure–function relationships.